The proposed research will investigate calpains and their inhibitor(s) in the retina and rod outer segments (ROSs). Calpains are calcium-activated neutral thiol proteases that are found in many tissues, where they are thought to play a regulatory role by specific and local proteolysis, particularly of cytoskeletal proteins. There is indirect evidence that calpains might play a role in the breakdown of outer segment organization during photoreceptor degeneration and/or disk detachment during normal daily shedding. In both cases, the most likely mechanism for calpain action would be by breaking down cytoskeletal protein(s). The present application proposes to characterize the calpains and their inhibitor(s), purified from retina and ROSs by column chromatography. It proposes to describe the localization of these enzymes and their inhibitor(s) under different normal and abnormal conditions, by light and electron immunocytochemistry, using antibodies made against the purified proteins. Finally it proposes to examine the distribution within the ROS of different cytoskeletal proteins, also by immunocytochemistry, with special emphasis on correlating it to the distribution of calpains. Major long-term objectives are to find out exactly what calpains do in the outer segment and to gain a better understanding of the outer segment cytoskeleton, particularly with respect to disk renewal and photoreceptor degeneration.